The invention relates to a chimeric poxvirus comprising the vector components of a retroviral (defective) genome. Furthermore, the invention relates to a chimeric poxvirus comprising the vector components and packaging components for producing retroviral defective viruses, as well as the use of the chimeric poxvirus according to the invention for gene or tumor therapy.
A central object of gene therapy is the stable genetic modification of target cells, and hitherto primarily viral vectors based on replication-deficient adenoviruses and retroviruses have been constructed for application in gene therapy.
The in vivo gene transfer with adenoviral vectors is very efficient, since particularly with adenoviral vectors, a large number of different cells can be transduced. However, in adenovirus infections, the DNA is not integrated into the host genome, but is present in episomal form. Moreover, a cellular immune reaction can be triggered which restricts the foreign gene expression (Li et al., 1993, Hum. Gene Ther. 4: 403-409). An attenuation of this reaction is possible by means of vectors in which the adenovirus E3 region has not been deleted (Ilan et al., 1997. Proc. Natl. Acad. Sci. USA 94: 2587‥2592). These modifications allow for a transient expression of the therapeutic gene lasting for months. A permanent action, however, cannot be obtained with adenovirus vectors, since, as stated above, these vectors are not integrated into the genome of the host cell in a stable manner.
In contrast thereto, retroviral vectors are capable of integrating foreign genes into the genome of the host cell in a stable manner. Retroviruses belong to the few presently known viruses which have mechanisms for integrating foreign DNA into the genome of the host cell and thus are capable of permanently transforming the latter. On account of this property, they are frequently used in gene therapy approaches. For this purpose, in techniques used so far, foreign genes have been cloned into the proviral DNA of retroviruses, wherein, however, replication-deficient retroviruses have been used, in which at least one of the retroviral genes encoding for the packaging function (gag-pol or env) has been inactivated or deleted. These constructs contain the packaging signal psi and the flanking Long Terminal Repeat (LTR) sequences of the provirus. These transcription units are introduced into the cells with the plasmid vectors by conventional transfection techniques and commonly transcribed in RNA in the nucleus under the control of the LTR promoter. On account of the packaging signal psi, such an RNA is accepted as genomic viral RNA and packaged in retroviral particles.
To form infectious retroviral particles, in the simplest case, the gene products gag-pol and env must be present. These genes can be inserted into the cells also without the packaging signal via plasmid transfection, or they may be provided via a helper virus. For preparing retroviral particles, also cell lines in which the viral genes for the packaging components gag, pol and/or env are integrated in a stable manner and which express the same, can be transduced. Such cell lines are termed xe2x80x9cpackagingxe2x80x9d lines and are described e.g. in WO 97/35996.
Plasmid constructs have been described, so-called xe2x80x9cplasmovirusesxe2x80x9d, which encode all the necessary components for the formation of non-replicating retroviral particles (Noguiez-Hellin et al. 1996, Proc. Natl. Acad. Sci. USA 93: 4175-4180).
The use of replication-deficient retroviruses does, however, not completely prevent the risk that the retroviral DNA recombines with the helper genes, whereby it may come to the production of replication-competent viruses which possibly may have a tumorigenic potential.
Although the transformation of cells with retroviral vectors is highly efficient in vitro, difficulties are encountered at present in gene-therapeutical applications. Thus, transfection techniques as are required in the production of retroviral vectors via plasmid constructs, can only be carried out in vitro. The direct administration of the plasmid constructs for production of the defective retroviral particles in the body is extremely inefficient. Likewise, retroviral particles in vitro can be produced only up to comparatively low titers. On account of their instability, concentration of the particles is possible also to a limited extent only. The low titers and the instability of retroviral particles thus frequently prevent the efficient transformation in vivo. Methods, such as the direct injection of the particles into the target organ, or after tissue removal, ex vivo-transformation and implantation of transformed cells are being attempted at present. Also, the targeted transduction of certain target organs, the so-called xe2x80x9ctargetingxe2x80x9d, is very difficult in case of retroviral vectors. It has been attempted to change the tropism of the particles by heterologous enveloping proteins (pseudotyping).
The wide-spread retroviral vectors based on simple retroviruses, such as, e.g., Moloney Murine Leukemia Virus (MLV), are able to transduce only cells in the division phase. Cell growth in many fully grown organs is, however, very low. In tests for hepatic gene therapy, it has thus been attempted i.a. to obtain growing and thus transformable tissue by a partial removal of the liver. Likewise, retroviral vectors based on complex retroviral viruses, such as, e.g., HIV (lentiviral vectors) have been constructed, since the latter can also transform non-dividing cells (Reiser et al., 1996. Proc. Natl. Acad. Sci. USA 93: 15266-15271).
To correct the defective genes in case of gene therapy, an efficient insertion of the corresponding genes as well as long-term expression of the genes are particularly important. New approaches for preparing vectors for the stable in vivo-transduction thus take advantage of the principle of using viruses as the carriers for retroviral defective particles. In doing so, particularly the large insertion potential with DNA viruses, such as herpes virus and adenovirus, are utilized. The furthest-developed approach of chimeric virus systems is the preparation of defective retroviral particles by co-infection of two recombinant adenoviruses which encode the retroviral vector and the packaging function, respectively, the retroviral vector and the packaging proteins being introduced via the adenovector system, whereby these cells become transient retroviral production cells, and the retroviral vector particles formed therewith can infect the neighbouring cells (Feng et al., 1997. Nat. Biotechn. 15:866-870, Bilbao et al., 1997. FASEB J. 11:624-634).
This system has, however, several decisive disadvantages: The accepting capacity of foreign genes of adenovirus vectors is restricted to a few kilobases so that it is difficult to unite all the functions for producing a defective retrovirus (packaging components and retroviral vector components) in one adenovirus. Such a construct is, however, the prerequisite for an efficient in vivo gene therapy. Moreover, both adenovirus and retroviral genes and genomes are transcribed in the nucleus of the host cell and replicated, respectively. This topographic vicinity makes recombination to wild type retroviruses probable. In principle, this system also does not allow for a deletion of important retroviral transcription control regions, since the latter are necessary for the transcription in the nucleus. Thus, the adenovirus/retrovirus system cannot be attenuated to the desired degree, in terms of safety technique. The same holds for the herpes simplex amplicon system; so far, the latter has only been described for the expression of retroviral structural proteins, wherein cells containing a lacZ provirus have been infected with an HSV amplicon vector containing the packaging components, and thus retroviral lacZ particles have been obtained (Savard et al., 1997. J. Virol. 71:4111-4117).
A disadvantage both with adenoviral and with retroviral vectors is in particular that introns for improving the foreign gene expression or for stabilising vector RNAs in the transduced cell cannot be applied since these introns have already been removed during the vector production by nucleus-specific splice mechanisms.
For a viral virus vector as a chimeric retrovirus carrier, also splicing and polyadenylating signals must be located at the correct site in the retroviral defective virus genome so as not to lead to a defective splicing or to a premature chain termination, respectively, during the transcription of the retroviral genomes to be transduced. If, for instance, the nuclear polyadenylating signal is put in front of the second, downstream retroviral LTR promoter, this will lead to a chain termination of the transcripts in the nucleus and to a retroviral defective genome no longer capable of transduction. In case of the atopic presence of these signals, the adeno- or herpes viruses replicating in the nucleus would not form a transducing defective retrovirus and cannot be made safer in this manner. The insertion of retroviral LTRs in herpes virus may, moreover, produce oncogenic subspecies from non-oncogenic herpes viruses (Isfort et al., 1992, Proc. Natl. Acad. Sci., USA 89;991-995). This is particularly possible because the life cycles of the herpes viruses are performed in the nucleus.
Also alpha viruses have been used as vectors for producing retroviruses. Coinfects of several Semliki Forest vectors obtained via RNAs synthesized in vitro resulted in infectious retroviral vector particles (Li et al., 1993. Hum. Gene Ther. 4:403-409). The natural tropism of carrier viruses could be used for the gene transfer into the respective preferred cell types and tissues.
It has been the object of the present invention to provide a vector system which does not have the above-mentioned disadvantages and allows for an efficient formation of retroviral particles.
According to the invention, this object has been achived by providing a chimeric poxvirus which comprises the sequences of the vector component of a retroviral particle.
By xe2x80x9cvector componentxe2x80x9d, in the present context a defective retroviral vector genome is understood which contains all the sequences necessary for the expression of the retroviral genome, including the packaging signal psi, as well as the sequences encoding a foreign protein. In the chimeric poxvirus of the invention, the sequence for the vector component in particular comprises a modified retroviral genome in which a foreign gene, in particular one (or several) sequence(s) encoding a foreign protein, antisense DNA, one (or several) ribozyme(s), are under the transcriptional control of a nucleus-active promoter, in particular a xcex2-actin, CMV or SV40 early promoter.
The foreign protein may be any desired protein, a protein for substitution therapy or tumor therapy being, however, particularly preferred. Proteins suitable for substitution therapy may be plasma proteins, such as, e.g., factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XIII, protein C, protein S, von Willebrand factor or erythropoetin. Proteins suitable for tumor therapy are tumor suppressor proteins, such as p53 or p73, or xe2x80x9csuicidal genesxe2x80x9d, such as HSV TK, immunostimulators, such as B7.
The poxvirus-, in particular the vaccinia virus transcription apparatus recognizes neither the LTR promoter of the retroviral provirus DNA as promoter, nor does it recognize the proviral RNA processing signals. Thus, according to a special aspect of the present invention, the sequences of the vector component are put under the transcriptional control of a poxvirus-specific promoter.
There, particularly preferred promoters are poxvirus promoters which control the expression of the early genes. With the chimeric vaccinia virus of the invention (Retrovac vector), these are particularly the early promoters which comprise both natural and synthetic promoters, as described, e.g., by Davison et al. (1989, J. Mol. Bio. 210:749-769) and Pfleiderer et al. (1995, Protein Expr. Purif. 6:559-569).
To prepare such constructs, e.g., the U3-region from the 5xe2x80x2 end of the proviral DNA can be deleted and replaced by a poxvirus-specific promoter. Likewise, the U5-region from the 3xe2x80x2 end of the proviral DNA can be deleted and a TTTTTNT signal can be added at the end of the xe2x80x9cRxe2x80x9d region. The repeated regions xe2x80x9cRxe2x80x9d at the ends of the transcripts are essential to the reverse transcription and thus to the functionality of the vector particles. Initiation upstream of the normal transcription start leads to the synthesis of a transcript having a non-repeated 5xe2x80x2 end, and thus, as expected, to reduced vector titers. With a view to a correct 5xe2x80x2 end of the RNA, the initiation site of the poxvirus promotor each inserted in the prototype construct should thus be optimized in each case. Optimisation of such constructs is within the general knowledge of the skilled artisan and can be carried out without great expenditures.
With the chimeric poxvirus according to the invention, defective retroviral particles can be prepared in a simple manner by infection of suitable packaging cell lines, such as, e.g., described in WO 97/35996, which express the packaging components.
According to a special embodiment of the present invention, the chimeric poxvirus contains the sequences which encode the vector components and the packaging components.
The chimeric poxvirus according to the invention, comprising both the vector component and the packaging component, is capable of liberating recombinant defective retroviruses in situ, since it expresses in the target cell both the genes for the packaging components and also a transcription unit which encodes a retroviral defective genome. Besides retroviral replication signals and packaging signals, the retroviral defective genome preferably also comprises the foreign gene to be transduced and expressed, which is controlled either by the promoter of the retroviral long terminal repeat (LTR) itself, or by a second promoter.
Within the present invention, by xe2x80x9cpackaging componentxe2x80x9d any genes necessary for forming a retroviral vector, such as gag-pol and env, of a retrovirus are understood. For the construction of the chimeric poxviruses of the invention (so-called RetroVac vectors), simple retroviruses, such as MLV, just as well as complex retroviruses, e.g. human immunodeficiency virus (HIV) can be used. Likewise, for changing the host spectrum, heterologous enveloping proteins (VSV-G, e.g.) can be expressed for the transforming retroviral particles, as has already previously been described for retroviral vectors. Generally, most of the further developments on retroviral vectors can also be applied to RetroVac vectors. This also includes the expression of foreign genes under the control of tissue-specific promoters and vectors for the site-specific integration of foreign DNA.
According to the invention, the gag, pol and env genes preferably are under the control of a poxvirus promoter. Preferred are promoters having a large early portion, in agreement with the expression characteristic of the poxvirus. The sequences gag/pol and env encoding the packaging components can be expressed in one transcription unit or on separate transcription units under the control of a poxvirus/vaccinia virus-specific promoter, wherein the transcription unit may be integrated in an essential or non-essential region of the poxvirus genome.
With poxvirus transcripts, splicing does not take place. Retroviral genes whose expression encompasses splicing, thus, according to a further aspect of the present invention, are cloned as intron-free reading frames into the chimeric poxvirus of the invention. Depending on the retroviral system selected, this aplies to the genes env, tat, ref or other ones.
With the chimeric virus according to the invention, defective retroviral particles can be produced in a simple manner both in vitro and in vivo. So far, no system has been described in which all the components for a defective retroviral particle have been encoded and combined on one single carrier virus. This is an essential pre-requisite for the efficient application of such vectors in vivo.
The development of packaging vectors and delivery/amplification systems of transducing defective retroviruses on the basis of chimeric poxviruses so far has not been described. What has been described, however, is the use of poxvirus vectors for the expression of retroviral components, in particular of gag-pol and env of HIV in poxvirus vectors (Moss, 1996, Proc. Natl. Acad. Sci., USA 93: 11341-11348, Paoletti, 1996, Proc. Natl. Acad. Sci., USA 93: 11349-11353). The purpose of these studies was, however, fundamental virological research and vaccine development. Thus, e.g., it is known that the expression of the HIV-1 gag-pol reading frame in VV (vaccinia virus) leads to the formation of pseudoparticles (Karacostas et al., 1989, Proc. Natl. Acad. Sci., USA 86:8964-8967). Likewise, the co-expression of gag and env leads to the formation of HIV-like particles which are particularly suitable for vaccine development (Haffar et al., 1990, J. Virol. 64: 2653-2659). Also the double-expression of gag-pol and env in poxvirus vectors as SIV candidate vaccine has been described (Hirsch et al., 1996, J. Virol. 70:3741-3752).
As the vectors for the chimeric poxviruses according to the invention, in particular chordopox viruses are used, which also include viruses from the group of the orthopox viruses and of the avipox viruses (Moss, 1996, Poxviridae: the viruses and their replication. In: Fields et al. (ed.) Fields Virology. Third Edition (3rd. Ed.) Vol. 2. Lippincott-Raven, Philadelphia, 2637-2671). Preferably, however, such poxviruses are used which infect mammalian cells, yet do not propagate therein (non-replicating vectors). As the vectors, thus preferably vaccinia viruses are used, in particular attenuated vaccinia viruses (Paoletti, 1996, Proc. Natl. Acad. Sci., USA 93: 11349-11353), Modified Vaccinia Ankara (MVA), or defective vaccinia viruses, such as described in WO 95/30018 and in Holzer et al. (1997, J. Virol, 71:4997-5002). The last-mentioned defective virus vector can easily be propagated to titers of 108 plaque forming units (PFU)/ml and concentrated to titers of 1011 PFU/ml. Moreover, e.g., D4-deleted defective viruses remain in the early phase of replication, which leads to a lasting synthesis of early RNAs. Only early vaccinia virus (VV)-RNAs have defined 5xe2x80x2 and 3xe2x80x2 ends, which is a basic pre-requisite for the synthesis of functional retroviral genoms. A vaccinia virus defective particle which expresses the entire genetic information for a retroviral vector causes the formation of non-replicating retroviral particles in the infected cell, which particles in turn have a transforming potential. Since, as stated above, particularly vaccinia viruses can be concentrated to titers of 1011 PFU/ml, the titer, based on the transformation (expressed in CFU/ml) is thus higher than conventional retrovirus titers ( greater than 106-107). By the expression of retroviral particles in situ, furthermore all the transforming particles released over time are relevant for the transduction and not, as in with the in vitro production of retroviruses, the transducing particles per volume of cell culture supernatant.
According to a special embodiment of the present invention, a non-replicating vaccinia virus, such as described in WO 95/30018, serves as the carrier of the genetic information for non-replicating defective retroviruses. Both the modified retroviral genome which contains the foreign gene and the packaging signal psi, and the retroviral genes gag-pol and env are encoded on the genome of a defective vaccinia virus. The sequences gag/pol and env encoding the packaging component can be expressed in one transcription unit or in several transcription units under the control of a poxvirus/vaccinia virus-specific promoter and can be integrated in an essential or non-essential region of the poxvirus genome. The sequences of the vector component can be inserted as an autonomous transcription unit, also under the control of a poxvirus, preferably an early poxvirus promoter, in an essential or non-essential region of the recombinant virus, insertion in an essential region being preferred.
The DNA sequence TTTTTNT leads to a termination of the early poxvirus/vaccinia transcription. To efficiently express the retroviral sequences, thus TTTTTNT signals possibly present in the retroviral sequences or in the foreign gene, respectively, should be modified by point mutation without changing the amino acid context of the retroviral proteins or destroying the controlling signals on the RNA genome of the defective retroviruses, such as e.g. psi, and the integration region xe2x80x9cattxe2x80x9d.
According to a special embodiment of the present invention, thus the sequences encoding the packaging components and the vector components, respectively, do not comprise any poxvirus/vaccinia virus-specific stop signals.
Just as for the expression of the vector component sequence, in the chimeric poxvirus-RetroVac system of the invention it is preferred to put the vector component sequence under the transcriptional control of a poxvirus-specific promoter.
In the preferred chimeric defective vaccinia virus constructs described in the present invention, downstream of the 3xe2x80x2 region a signal was inserted for the termination of early vaccinia virus transcripts, and thus the 3xe2x80x2 end of the mRNAs forming differ from that of previously described retroviral vector genomes. Only with the so-called early vaccinia mRNAs, most of them have a defined 3xe2x80x2 end. Late vaccinia mRNAs which constitute the major portion of the viral transcripts do not terminate at concrete signals, have heterogenous lengths and thus are not suitable as retroviral vector genomes. D4-defective VV do not enter the late phase of replication. This could be demonstrated by 35S labelling experiments of proteins and in Northern Blot experiments by an unexpectedly long lasting early expression (Holzer et al., 1997, J. Virol. 71:4997-5002). Thus they proved to be a tool unique among poxviruses, for the synthesis of defined retroviral genomes.
One advantage of the chimeric poxviruses according to the invention is that they can be propagated and concentrated to very high virus titers. Moreover, the vectors derived from poxvirus, in particular vaccinia virus, are extremely stable, efficiently infect organs, such as liver or spleen, and produce transforming, yet not replicating, retroviral particles directly in the target organ (in vivo amplification of the retroviral particles).
Since poxviruses are tissue-specific (primary affine organs), a transformation is effected by the chimeric poxvirus of the invention, in particular the chimeric vaccinia virus, in a tissue-specific manner. For instance, the infection with the chimeric vaccinia virus (RetroVac hybrid vectors) preferably takes place in those tissues which correspond to the tropism of vaccinia virus, and which also express the receptors for the retroviral enveloping proteins used. Via suitable combinations of the vaccinia virus strain used and pseudotyping, a more stringent targeting of both the therapeutic gene and of the retroviral vectors can be achieved.
The chimeric poxvirus vectors, in particular the RetroVac vectors based on defective vaccinia virus, combine the ability of retroviruses to integrate foreign DNA in target cells in a stable manner with the technical advantages of poxvirus/vaccinia vectors. A main characteristic of the system is that when a host cell is infected by the chimeric poxvirus, in particular a RetroVac vector based on a vaccinia virus vector, the proteins necessary for the formation of functional retroviral particles are expressed and the mRNAs containing the foreign genes are transcribed and packaged as genomic RNA in retroviral particles. These particles are not capable of propagating (replication-deficient), yet they do have a transforming potential. While the cells primarily infected with the chimeric poxvirus, in particular with the chimeric vaccinia virus, will die, secondary retroviral infection of further cells by the retroviral particles formed will lead to a permanent integration of the retroviral sequences, and thus of the sequences encoding the foreign gene, into the cell genome.
The liberation of defective retroviral particles by the chimeric poxviruses of the invention has been surprising insofar as normally the transcription of retroviruses as well as the capping of the genomic RNAs occur in the nucleus (Coffin, 1996, Retroviridae: the viruses and their replication. In: Fields et al. (ed.) Fields Virology. Third Edition (3rd Ed.) Vol. 2, Lippincott-Raven, Philadelphia, 1767-1847). In the chimeric poxvirus system shown, transcription and capping occur in the cytoplasm. The vaccinia virus-specific cap structures thus are no obstacle for a retroviral packaging. Surprisingly it has been found that transcripts generated by the cytoplasmatic poxvirus/vaccinia transcription system are compatible with the retroviral transcription and replication system. This was unexpected insofar as the polyadenylating signals which are recognized in the nucleus are located within the U3 and R regions of the provirus.
As a rule, poxviruses are lytic viruses, whereby cells which have been infected by a chimeric poxvirus will die in most instances. On account of the lesions thus caused in the tissue, division of neighbouring cells can becaused which thus become even more susceptible to retroviral transformation by the retroviral particles formed. In case of ectodermal cells, this proliferation effect is increased by growth factors of the poxvirus itself.
In a special embodiment, thus the chimeric poxvirus according to the invention comprises sequences encoding a growth factor or a mitogen. By this, the proliferation effect of the neighbouring cells of the poxvirus-infected cells can be increased by the expression of chimeric poxvirus.
In a further special embodiment, it is possible to construct RetroVac vectors on the basis of lentiviruses, in which, however, accessory lentivirus genes must be expressed in the VV carrier, which give the system the properties desired.
In contrast to plasmid transfection, chimeric adenovirus/retrovirus vectors or chimeric herpes virus/retrovirus vectors, expression by vaccinia virus occurs in the cytoplasm of the host cell. While after a plasmid transfection or in case of an adenovirus/retrovirus infection the transcription of the foreign genes occurs in the nucleus by aid of the cellulary transcription apparatus, the gene expression of vaccinia virus exclusively occurs in the cytosol, by means of a viral transcription apparatus different from the cellular one. This constitutes a decisive safety advantage of the poxvirus vector of the invention insofar as by this the generation of replicating retroviruses becomes very unlikely. This approach for the first time allows for the arrangement of nuclear transcription signals on the retroviral genome exclusively according to the point of view of optimal foreign gene expression and of the safety in the transduced target cell, since the transcription of genomic retroviral RNA by vaccinia virus occurs independently of the nucleus of the target cell. Likewise, introns can be used to enhance the expression of a therapeutic gene, since the former do not influence vector RNA expression in the vaccinia virus system.
A further particular advantage of the virus vector system of the invention based on a DNA virus replicating in the cytoplasm is that, contrary to viruses propagating in the nucleus, retroviral transcription units may contain transcription signals which normally are not allowed or not possible in connection with the nucleus, since splicing does not occur in the life cycle of a cytoplasmatically transcribing virus vector. Thus, in the RetroVac system, e.g., the retroviral packaging signal psi can be flanked by splicing signals, which, after transduction of the retroviral defective genomes and transcription of the same in the host will lead to RNAs which have no packaging signals and thus have lost an essential feature of retroviral genomes (cf. FIG. 2C). This possibility substantially increases the safety of the system of the retroviral defective genomes produced in the poxvirus system. The functionality of the packaging signal outside of the wild type context has already been shown.
A further aspect of the present invention is that in case of a cytoplasmatic transcription of the retroviral defective RNAs, established transcription units having intron-exon structure can be integrated undamaged (without splicing) into the genome of the host via retroviral transduction (cf. FIG. 2A). When establishing permanently expressing cell lines, as a rule gene cassettes are transferred, consisting of promoter, open reading frame (ORF) of the foreign gene, intron and polyadenylating signal, cloned into a bacterial plasmid. For an optimal mRNA production, introns are required in transcription units, in particular this has also been observed in transgenic animals. Most of the commercially available expression vectors for higher cells contain empirically determined promoter-intron combinations within their expression cassettes; e.g., the CMV promoter/enhancer SV40 intron combination in vector pCMVxcex2 (Clontech Laboratories, Palo Alto) has proved successful. Such optimized units having intron-exon structure cannot be transduced via the hitherto known retroviral or chimeric, respectively, gene transfer vectors.
The RetroVac system according to the invention solves this problem, since on account of the missing splicing apparatus, no introns are removed in the vaccinia virus during the transcription. After transduction of the retroviral defective genome produced in the RetroVac system, the complete transcription unit thus can be integrated in the target cell (cf. FIG. 2A). This is particularly important at the transfer of cDNAs difficult to be expressed for the purpose of gene therapy, such as those of the coagulation factor VIII, and allows for the transfer of optimized promoter/intron combinations.
According to a special aspect of the invention, thus by the system according to the invention defective retroviral particles comprising an intron-containing genome are provided.
An RNA processing signal which may be transferred into the target cell at a defined site merely by cytosolic transcription systems is an internal polyadenylating signal which causes a defined termination of the transcript in the nucleus (FIG. 2B). Termination of the transcript before completion of a complete retroviral defective genome in the nucleus, such as by transduction of retroviral defective genomes produced in the RetroVac system, allows for a particularly safe gene therapy, since the foreign gene once integrated cannot be transduced further, because its transcript terminates prematurely. Likewise, the localisation of the packaging signal between splice signals (FIG. 2C) is possible only in the RetroVac system, which, in the target cell, leads to the transcription of retroviral defective genomes which lack the packaging signal. Thus, in combination with internal polyadenylating signals, particularly safe gene cassettes can be transferred.
A further aspect of the present invention relates to a composition comprising a chimeric poxvirus of the above-defined type and a pharmaceutical carrier.
The RetroVac system according to the invention can be utilized in all applications for which a gene therapy with retroviruses is meaningful. It is suitable for the in vivo gene therapy for the treatment of plasma protein defects, in particular hemophilias (factor IX and factor VIII deficiencies) and erythropoetin deficiency. Administration may be intravenously or intramuscularly.
With an appropriate formulation, the stability of the chimeric poxviruses according to the invention also allows for an oral administration of the vectors as sprays, which enables inhalation treatment of cystic fibrosis.
A further application of the RetroVac vectors is tumor therapy. It has been shown, for instance, that the transfer of so-called suicide genes into tumors or their metastases has proven to be promising in animal experimental models (Caruso et al., 1993, Proc. Natl. Acad. Sci., USA 90: 7024-7028, Culver et al., 1992, Science 256:1550-1552). In such experiments, the HSV-TK gene which converts non-toxic nucleoside analogs (such as Ganciclovir) into toxic ones, was transduced by intratumoral injection of packaging cell lines which in situ liberate defective retroviral particles, and subsequently a chemotherapy was carried out with Ganciclovir. With the RetroVac vector of the invention, thus suicide genes can safely and more efficiently be administered.
Likewise, apoptosis-induced tumor-suppressor genes, such as, e.g., the p53 gene which is altered in more than 50% of the tumors, can be inserted as foreign gene in the RetroVac vector of the invention. At the gene-therapeutical transfer of these tumor suppressor genes, the tumor cells would stop their growth on account of the endogenous control.
An ex vivo cell transduction for tumor therapy (e.g. leukemia) likewise is possible; furthermore, the direct intratumoral injection for cancer therapy.
To increase the safety of the system, vectors can be used which carry the so-called suicide genes, such as, e.g., the herpes simplex virus (HSV) with an inserted thymidine-kinase gene, which would allow for a chemotherapy, if there were an activation of oncogens in vivo due to the transduction procedure.
The system can also be used for producing permanent cell lines, since retroviral particles allow for a particularly efficient transduction and since on account of the transfer of RNA processing signals (introns, polyadenylating sites) optimal gene cassettes can be transferred. In this manner, the highly efficient transfer of gene cassettes has been possible for the first time, which in connection with screening techniques allows for the rapid identification of highly expressing cell clones.
A special aspect of the present invention thus relates to the use of the chimeric poxvirus according to the invention for producing a medicament, which in particular can be used for gene therapy and tumor therapy.
Within the scope of the present invention, defective retroviral particles were obtained via the above-mentioned chimeric poxviruses, which are particularly characterized in that they still comprise an intron-containing genome.